Quantitative analysis, particularly the automated quantitative analysis of cytological, hematological and histological specimens requires exacting control of dyes, dye solutions and staining processes. Quantitative measurements used to differentiate normal from pathologic specimens may be expressed either as light transmission, integrated optical densities, ploidy, light scattering, light polarization effects and fluorescence. Since these measurements are strongly dependent on the staining process, a characterization and standardization of the dyes and staining solutions used is necessary. The conventional methods of standardizing and evaluating dyes and staining solutions include the physical and chemical characterization of the dyes and also the direct visual assessment of stain performance.
However, the physical-chemical approach of quantitating the dye and its concentration does not provide any information on the specimen-dye interactions and cannot measure changes in the process, as the dyeing or staining baths which are used are exposed to different conditions.
The visual assessment of stain performance suffers from the lack of a reproducible specimen by which the performance can be quantitatively evaluated. Non-standardized specimens can only provide a qualitative analysis of the process and lack the necessary reproducibility to provide a quantitative analysis.
The difficulty of comparing biological samples quantitatively is due in large part to variations in the staining process. Dyes vary from batch to batch, and their solutions may vary over time for the same batch or even for the same preparation. This variability is especially true for natural dyes; and the Papanicolaou stain, although very useful for cytology, is difficult to standardize. This shortcoming is particularly problematic for automated analysis.
Even such promising digital parameters as cytoplasmic staining intensity, ploidy and nuclear integrated optical density, are strongly influenced by variations in the staining process. Cytology smears can vary by as much as 10-23%, mostly due to specimen processing and staining. A better understanding and control of dyes and staining processes is needed before automated methods of cytologic screening can be widely applied.
Therefore a need exists for a standard specimen which is reproducible to high degree of precision. Each sample must react in the same way with the staining solution and must stain uniformly so that light transmission does not vary for different microspectrophotometric fields.
Specimens having slight variations in the concentration of biological molecules which bind the dyes, or having any variation in thickness, do not meet the necessary criteria, since the transmitted light varies in accordance with the amount of dye bound along the light path. For these reasons, naturally occurring biological objects, even the most carefully controlled cell cultures, cannot therefore be used.
A standard specimen, to be useful, must be composed of molecules similar to those which bind dyes in the biological objects of interest, but must be strictly controlled so that a number of samples subjected to the same staining solutions at the same time will produce equivalent microspectrophotometric measurements to a high degree of precision. This must be true for different fields in the same specimen and different specimens.
The instant invention meets these needs, providing a standard specimen which can calibrate stain uptake and define a quantitative baseline for sample comparison. The inventive specimen utilizes extracts of biological preparations containing proteins and/or nucleic acids, which mimic the staining characteristics of biological specimens. These extracts are incorporated into a reproducible matrix which can be sectioned to a controlled thickness. This ensures the production of a large number of samples with almost identical capability for dye binding.
One of the advantages obtained by using the standard specimen according to the invention is that the standard specimen can quantitate the stain performance of stains, staining solutions, and staining processes used on biological substrates and can calibrate the process, under any conditions, regardless of the dye used or its concentration or the conditions to which it is exposed. The standard specimen will thus allow detailed quantitation and evaluation of the dyeing or staining process, thereby providing information for process control and quantitative analysis comparisons.